System and method for controlling outlet flow of a device for separating cellular suspensions

ABSTRACT

A system for separating a suspension of biological cells is disclosed comprising a single-use fluid circuit and a durable hardware component. The fluid circuit comprises a separator having a housing that includes an inlet for introducing the suspension of biological cells into the gap, a first outlet in communication with the gap for flowing a first type of cells from the separator, and a second outlet in communication with the second side of the filter membrane for flowing a second type of cells from the separator. The hardware component comprises a pump for flowing the suspension of biological cells to the inlet of the separator and at least one flow control device associated with the first outlet and the second outlet of the separator for selectively opening and closing the outlets so as to permit one of the first type of cells and the second type of cells to flow out of the separator in accordance with a predetermined duty cycle equal to the ratio of a target flow rate of first type of cells through the first outlet to the predetermined inlet flow rate.

FIELD OF THE INVENTION

The present disclosure is directed to methods for controlling flowthrough a device for the separation of biological cells in a suspensionusing a pulse width modulation technique, and to and systems employingsuch methods. The method controls the outlet flow rate from theseparator by alternating between an open and closed first outlet line,with a second outlet line being open or closed opposite to the firstoutlet line. Modulation of the ratio of time that the first outlet lineis open to the time that the first outlet line is closed defines a dutycycle that determines the outlet flow rates through both the first andsecond outlet lines. In a specific example, the separator comprises amembrane separation system and the method controls the fluid flowing outof the first outlet line (through which the retentate flows) and thesecond outlet line (through which the filtrate flows) using the pulsewidth modulation technique.

BACKGROUND

The use of devices for the separation of whole blood or into itsconstituent components is widespread. Such devices commonly utilizecentrifuges (that separate the cellular components based on theirdensity) or filter membranes (that separate the cellular componentsbased upon their size).

Typically, the control of fluid into and out of a centrifugal orspinning membrane separation device has been accomplished by applying afirst pump to the inlet line of the separator to supply a blood source,and a second pump applied to either a first outlet line (for theretentate, in the case of a spinning membrane separator) or a secondoutlet line (for the filtrate in the case of a spinning membraneseparator) to control the flow of fluid through the membrane.

In the case of a spinning membrane separator, to force a fluid to flowacross a membrane a gradient must be formed across the membrane. For aspinning membrane separator, a pressure gradient, commonly referred toas the transmembrane pressure (TMP), is generated to force fluid(filtrate) to flow through the membrane while particles or cells(retentate) greater than the membrane pore size are retained. See, e.g.,US 2013/0345674, which is incorporated herein by reference. For example,if the inlet pump is pumping at 50 ml/min and the retentate pump inpumping at 30 ml/min, a TMP will be produced and filtrate will flowthrough the membrane at 20 ml/min (difference between inlet andretentate rates).

While the use of a pump on each of the inlet line and outlet line(s) ofthe separation system has proven to be effective for many applications,there is a desire to simplify the required hardware and to reduce thesize of the system.

SUMMARY

By way of the present disclosure, a cellular separation system andmethod are provided in which the pump associated with one of the outletsof the separator is eliminated, and an automated clamp or stopcock isinstead associated with each of a first and second outlet line forcontrolling flow out of the separator. Actuation of the clamps/stopcocksto alternately open and close the two outlet lines in opposition to eachother permits the use of a pulse width modulation (PWM) technique tocontrol the outlet flow from the separator. Opening and closing the twooutlet lines in opposition for specific time durations permits theachievement of a specific average flow rate out of the separator.

In a first aspect, a system for separating a suspension of biologicalcells is provided comprising a durable hardware component and a singleuse fluid circuit. The fluid circuit comprises a separator having ahousing with an inlet for introducing the suspension of biological cellsinto the separator, a first outlet in communication with the separatorfor flowing a first type of cells from the separator and a second outletin communication with separator for flowing a second type of cells fromthe separator, and a hardware component comprising a pump for flowingthe suspension of biological cells to the inlet of the separator, atleast one flow control device associated with the first outlet and thesecond outlet of the separator for selectively opening and closing thefirst and second outlets so as to permit one of the first type of cellsand the second type of cells to flow out of the separator.

In a specific example, the fluid circuit comprises a spinning membraneseparator having a housing and a relatively-rotatable filter membranehaving a first side and a second side. A gap is defined between thehousing and the first side of the filter membrane, while a flow path isprovided that is in fluid communication with the second side of thefilter membrane. The housing includes an inlet for introducing thesuspension of biological cells into the gap, a first outlet incommunication with the gap for flowing the first type of cells from theseparator, and a second outlet in communication with the second side ofthe filter membrane for flowing the second type of cells from theseparator.

The hardware component comprises a pump for flowing the suspension ofbiological cells to the inlet of the separator. At least one flowcontrol device is associated with the first outlet and the second outletof the separator for selectively opening and closing the first andsecond outlets so as to permit one of the first and second types ofcells to flow out of the separator. The flow control device(s) maycomprise a clamp for each outlet line from the separator. Alternatively,the flow control device may comprise a two-way stopcock that connects toboth of the two outlet lines from the separator.

The hardware component further comprises a programmable controllerconfigured to operate the pump so as to flow the suspension ofbiological cells to the inlet of the separator at a predetermined inletflow rate and to alternately open and close the flow control device inaccordance with a predetermined duty cycle. More specifically, theprogrammable controller is configured to alternately open and close theflow control device(s) such that the duty cycle is equal to the ratio ofa target flow rate of the first type of cells through the first outletto the predetermined inlet flow rate.

In a second aspect, the programmable controller is further configured todetermine the target flow rate of first type of cells as the product theinlet flow rate times the ratio of cell concentration of the suspensionof biological cells to a target cell concentration of the first type ofcells.

In a third aspect, the programmable controller is further configured toalternately open and close the flow control device at a predeterminedfrequency.

In a fourth aspect, the programmable controller is further configured toestablish the predetermined frequency for the duty cycle based on theconcentration of cells in the suspension of biological cells beingseparated.

In a fifth aspect, the predetermined frequency for the duty cycle isdirectly proportional to the concentration of cells in the suspension ofbiological cells being separated.

In a sixth aspect, a method for separating a suspension of biologicalcells is provided using a system as described above, comprisingoperating the pump so as to flow the suspension of biological cells tothe inlet of the separator at a predetermined inlet flow rate, andalternately opening and closing the flow control device(s) in accordancewith a predetermined duty cycle. More specifically, the method comprisesalternately opening and closing the flow control device(s) such that theduty cycle is equal to the ratio of a target flow rate of a first typeof cells through the first outlet to the predetermined inlet flow rate.

In a seventh aspect, the method comprises determining the target flowrate of the first type of cells as the product the inlet flow rate timesthe ratio of cell concentration of the suspension of biological cells toa target cell concentration of the first type of cells.

In an eighth aspect, the method comprises alternately opening andclosing the flow control device at a predetermined frequency.

In a ninth aspect, the method comprises establishing the predeterminedfrequency for the duty cycle based on the concentration of cells in thesuspension of biological cells being separated.

In a tenth aspect, the predetermined frequency for the duty cycle isdirectly proportional to the concentration of cells in the suspension ofbiological cells being separated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic cross-sectional view of a spinning membraneseparator of the type that may be advantageously used in the system andmethod of the present disclosure.

FIGS. 2A and 2B are schematic representations of a system in accordancewith the present disclosure in which each of the outlet lines from theseparator has a flow control device in the form of a clamp with thefirst outlet line or retentate line open/second outlet line or filtrateline closed (FIG. 2A) and the retentate line closed/filtrate line open(FIG. 2B).

FIGS. 3A and 3B are schematic representations of a system in accordancewith the present disclosure in which both of the outlet lines from theseparator share a common flow control device in the form of atwo-position stopcock with the retentate line open/filtrate line closed(FIG. 3A) and the retentate line closed/filtrate line open (FIG. 3B).

FIGS. 4-6 are plots of retentate rate vs. time for duty cycles of 50%,10% and 80%, respectively, showing the average retentate flow rate undersuch conditions.

DETAILED DESCRIPTION

A more detailed description of the systems and methods in accordancewith the present disclosure is set forth below. It should be understoodthat the description below of specific devices and methods is intendedto be exemplary, and not exhaustive of all possible variations orapplications. Thus, the scope of the disclosure is not intended to belimiting, and should be understood to encompass variations orembodiments that would occur to persons of ordinary skill.

While the following describes the method in the context of system thatutilizes a spinning membrane separator, the method is equally applicableto systems that use other types of separation devices, such ascentrifuges. Terms that relate specifically to membrane filtration, suchas “retentate” and “filtrate”, are understood to have counterparts incentrifugal separation. Thus “retentate” should be broadly understood torefer to a first type of blood cell in the suspension, while “filtrate”should be understood to refer to a second type of blood cell in thesuspension and/or a non-cellular fluid (e.g., plasma) in which the bloodcells are suspended. Similarly, in the context of unspecified types ofseparators, reference to “a second type of cells” should be understoodto include a second type of blood cell in the suspension and/or thenon-cellular fluid in which the blood cells are suspended.

Turning to the FIGS. 1-3, a system 10 for separating a suspension ofbiological cells is provided comprising a durable hardware component 12and a single use fluid circuit 14. The fluid circuit 14 comprises aspinning membrane separator 16 (best seen in FIG. 1) having a housing 18and a relatively-rotable filter membrane 20 having a first side and asecond side. A gap 22 is defined between the housing 18 and the firstside of the filter membrane, while a flow path 24 is provided that is influid communication with the second side of the filter membrane 20.

The housing 18 includes an inlet 26 for introducing the suspension ofbiological cells into the gap 22 to which a source of the biologicalsuspension to be separated (reservoir 28) is connected by a first tubingsegment 30. While the suspension to be separated is shown as beingcontained in a reservoir 28, it could also be sourced directly from adonor by means of a donor access device (such as a phlebotomy needle) onthe free end of the first tubing segment 30.

The housing 18 further includes a first outlet 32 in communication withthe gap 22 for flowing retentate from the separator 16 through a secondtubing segment 34 to a first collection container 36, and a secondoutlet 38 in communication with the flow path 24 on the second side ofthe filter membrane for flowing filtrate from the separator through athird tubing 40 segment to a second collection container 42. In oneexample, the suspension of biological cells may be whole blood, theretentate may be red blood cells and the filtrate may be plasma. In asecond example, the suspension of biological cells may be platelet richplasma, the retentate may be a platelet concentrate and the filtrate maybe platelet free plasma.

The hardware component 12 comprises a pump 44 that cooperatively engagesthe first tubing segment 30 for flowing the suspension of biologicalcells to the inlet 26 of the separator 16. At least one flow controldevice is associated with each of the second and third tubing segments34, 40, that are in fluid communication with the first outlet and thesecond outlets 32, 38, respectively, of the separator 16. These flowcontrol devices selectively alternately open and close the second andthird tubing segments so as to permit one of the retentate and thefiltrate to flow out of the separator through the first and secondoutlets. With reference to the embodiment of FIGS. 2A and 2B, the flowcontrol devices comprise clamps 46, 48 respectively associated thesecond and third tubing segments 34, 40. With reference to theembodiment of FIGS. 3A and 3B, the flow control device comprises atwo-way stopcock 50 that is connected to both the second and thirdtubing segments 34, 40.

The hardware component 12 further comprises a programmable controller 52configured to operate the pump 44 so as to flow the suspension ofbiological cells to the inlet 26 of the separator 16 at a predeterminedinlet flow rate, and to alternately open and close the flow controldevice(s) 46, 48, 50 in accordance with a predetermined duty cycle. Morespecifically, the programmable controller 52 is configured toalternately open and close the flow control device(s) 46, 48, 50 suchthat the duty cycle is equal to the ratio of a target flow rate ofretentate through the first outlet to the predetermined inlet flow rate.As illustrated, the hardware component also includes a pressure sensor54 for measuring the fluid pressure at the inlet 26 of the separator 16.

As noted above, the flow of fluid out of the spinning membrane separator16 is controlled by applying a pulse width modulation (PWM) technique.Thus, fluid flow through the spinner gap 22 and across the spinningmembrane 20 is accomplished by using a single pump 44 to control theinlet rate, and PWM, rather than second pump, is used to control theoutlet rate.

Specifically, the flow rate of retentate and filtrate out of theseparator is controlled by alternating or pulsing between an open andclosed retentate line 34, with the filtrate line 40 being open or closedin opposition to the retentate line 34 (i.e., if retentate line 34 isclosed, filtrate line 40 must be open, and vice versa). The alternatingof the open and closed states for the retentate and filtrate lines isillustrated in FIGS. 2A and 2B, in which each of the retentate andfiltrate lines 34, 40 has a clamp 46, 48 associated therewith (with an“X” through the clamp indicating that the clamp has closed itsassociated tubing segment), and in FIGS. 3A and 3B, in which theretentate and filtrate lines 34, 40 share a common two-position stopcock50. Modulation of the “retentate line open” pulse width (i.e., changinghow long the retentate line is open vs. closed) allows for the retentaterate (and filtrate rate) to be changed and controlled. Alternating theflow control devices between open and closed states, with a specificduty cycle, will lead to an “average” output flow rate somewhere inbetween the flow rates obtained in the open and closed states.

For example, and presuming that the retentate and filtrate clamps 46 and48 are in opposing states, if the inlet pump is flowing at 50 ml/min andthe retentate line is closed, the flow through the retentate line is theequal to 0 ml/min, but if the retentate line is open, flow through theline is 50 ml/min. This scenario is depicted in FIG. 4, in which theretentate line opens and closes with a duty cycle of 50% to produce anaverage retentate flow rate of 25 ml/min. If the retentate linealternates between open and closed states is sufficiently fast relativeto the mechanical compliance of the tubing kit, the average output ratewill be substantially constant over time.

The duty cycle, or percentage of time the retentate line is open,required to achieve a targeted retentate flow rate is equal to the ratioof the target retentate rate to the constant inlet pump rate.

${{Duty}\mspace{14mu} {Cycle}} = \frac{{Target}\mspace{14mu} {Retentate}\mspace{14mu} {Rate}}{{Inlet}\mspace{14mu} {Rate}}$

To determine the amount of time which the retentate line should beopen/closed over a specific time period to achieve the targeted rate,the Duty Cycle calculated above can be divided by the desired frequency(how often the flow control devices are to be opened and closed)according to the definition of a duty cycle:

${{Duty}\mspace{14mu} {Cycle}} = {\frac{{Time}\mspace{14mu} {Active}}{{Signal}\mspace{14mu} {Time}\mspace{14mu} {Period}} = \frac{{Time}\mspace{14mu} {Retentate}\mspace{14mu} {Line}\mspace{14mu} {Open}}{{Signal}\mspace{14mu} {Time}\mspace{14mu} {Period}}}$${{Where}\text{:}\mspace{14mu} {Signal}\mspace{14mu} {Time}\mspace{14mu} {Period}} = \frac{1}{Frequency}$${{Time}\mspace{14mu} {Retentate}\mspace{14mu} {Line}\mspace{14mu} {Open}} = \frac{{Duty}\mspace{14mu} {Cycle}}{Frequency}$Time  Retentate  Line  Closed = Signal  Time  Period − Time  Retentate  Line  Open

The duty cycle is dependent only on the desired retentate flow rate fora given inlet rate. Thus, the retentate flow rate has the potential tobe any value in practice, and will vary depending on target retentaterates unique to each application. Inlet rates are also dependent on theapplication.

Retentate rates are selected to achieve a targeted outlet concentrationbased on a known inlet concentration of cells according to the followingrelationship: Retentate Rate=(Inlet Rate×Inlet Cell Concentration[HCT])/Target Outlet Cell Concentration [HCT]. For example, in wholeblood filtration in which the inlet flow rate is 50 ml/min and the bloodis 40% HCT, if a target of 80% retentate HCT is desired, the retentaterate is equal to (50 ×40)/80=25 ml/min.

Applications such as cell washing or platelet rich plasma volumereduction will tend to have lower duty cycles, as the retentate flowrates are typically very low compared to inlet flow rates. Applicationssuch as whole blood filtration or plasmapheresis would apply higher dutycycles, as the retentate rate is around half of the inlet rate. Forexample, platelet rich plasma volume reduction application may apply aninlet flow rate around 25 ml/min and target a retentate rate of 5ml/min, leading to a duty cycle of 20%. On the other hand, whole bloodfiltration may apply an inlet flow rate of 50 ml//min, and target aretentate rate around 25 ml/min, leading to a duty cycle of 50%. Dutycycles of as high as 80-90%, may be applied during priming applications,or during procedures as well.

The duty cycle is selected regardless of how it affects thetransmembrane pressure (TMP) of the spinning membrane separator.However, a particular duty cycle can have an impact on the measured TMPif the correct frequency is not chosen. For example, a duty cycle of 50%at a frequency of 1 Hz may cause the measured TMP to oscillate, sincethe retentate line is open/closed relatively slowly, whereas a dutycycle of 50% with an outlet line open/close frequency of 10 Hz may pulsefast enough for the measured TMP to be smooth over time. Thus, asufficiently high frequency is selected that, regardless of the dutycycle, the flow is no more pulsatile, and potentially less pulsatile,than when the flow is controlled by a pump associated with one of theoutlet lines. This, in turn, will cause the measured TMP to be no morepulsatile than that experienced when a pump is used to control theoutput flow rate.

The preferred frequency will likely vary depending on application. Ingeneral, applications involving red blood cells (like whole bloodfiltration or plasmapheresis) will likely utilize higher frequenciesthan applications involving only platelets or white blood cells (likecell washing), as the overall cell volume of whole blood filtrationprocedures is significantly higher than that of cell washing procedures,which would result in membrane fouling and hemolysis if outlet line isnot pulsed at a sufficiently high frequency.

EXAMPLES FOLLOW Example 1 (FIG. 5): Inlet Rate=50 Ml/Min, TargetRetentate Rate=5 Ml/Min, Frequency 10 Hz

${{Duty}\mspace{14mu} {Cycle}} = {\frac{{Target}\mspace{14mu} {Retentate}\mspace{14mu} {Rate}}{{Inlet}\mspace{14mu} {Rate}} = {\frac{5}{50} = 0.1}}$${{Signal}\mspace{14mu} {Time}\mspace{14mu} {Period}} = {\frac{1}{Frequency} = {\frac{1}{10\mspace{14mu} {Hz}} = {0.1\mspace{14mu} \sec}}}$${{Time}\mspace{14mu} {Retentate}\mspace{14mu} {Line}\mspace{14mu} {Open}} = {\frac{{Duty}\mspace{14mu} {Cycle}}{Frequency} = {\frac{0.1}{10} = {0.01\mspace{14mu} \sec}}}$Time  Retentate  Line  Closed = Signal  Time  Period − Time  Retentate  Line  Open = 0.1  sec  − 0.01  sec  = 0.09  sec 

Example 2 (FIG. 6): Inlet rate=50 Ml.Min, Target Retentate Rate=40Ml/Min, Frequency 10 Hz

${{Duty}\mspace{14mu} {Cycle}} = {\frac{{Target}\mspace{14mu} {Retentate}\mspace{14mu} {Rate}}{{Inlet}\mspace{14mu} {Rate}} = {\frac{40}{50} = 0.8}}$${{Signal}\mspace{14mu} {Time}\mspace{14mu} {Period}} = {\frac{1}{Frequency} = {\frac{1}{10\mspace{14mu} {Hz}} = {0.1\mspace{14mu} \sec}}}$${{Time}\mspace{14mu} {Retentate}\mspace{14mu} {Line}\mspace{14mu} {Open}} = {\frac{{Duty}\mspace{14mu} {Cycle}}{Frequency} = {\frac{0.8}{10} = {0.08\mspace{14mu} \sec}}}$Time  Retentate  Line  Closed = Signal  Time  Period − Time  Retentate  Line  Open = 0.1  sec  − 0.08  sec  = 0.02  sec 

Thus, an improved spinning membrane separation system and method havebeen provided in which the number of components has been reduced overprior systems, thus facilitating simplification of the design andreduction is size for the system. The system and method should alsoprovide for greater accuracy in controlling the outlet flow from theseparator, as discrepancies between the target flow rate and actual flowrate through peristaltic pumps due to, e.g., pump inlet and outletpressures, tubing dimensions and tolerances, and pump motor rotationerrors, will no longer affect the outlet flow from the separator.

1. A method for separating a suspension of biological cells using asystem comprising a fluid circuit comprising a separator having ahousing with an inlet for introducing the suspension of biological cellsinto the separator, a first outlet in communication with the separatorfor flowing a first type of cells from the separator and a second outletin communication with separator for flowing a second type of cells fromthe separator, and a hardware component comprising a pump for flowingthe suspension of biological cells to the inlet of the separator, atleast one flow control device associated with the first outlet and thesecond outlet of the separator for selectively opening and closing thefirst and second outlets so as to permit one of the first type of cellsand the second type of cells to flow out of the separator, the methodcomprising: a) operating the pump so as to flow the suspension ofbiological cells to the inlet of the separator at a predetermined inletflow rate; and b) alternately opening and closing the flow controldevice in accordance with a predetermined duty cycle.
 2. The method ofclaim 1 further comprising alternately opening and closing the flowcontrol device such that the duty cycle is equal to the ratio of atarget flow rate of the first type of cells through the first outlet tothe predetermined inlet flow rate.
 3. The method of claim 2 furthercomprising determining the target flow rate of first type of cells asthe product of the inlet flow rate times the ratio of cell concentrationof the suspension of biological cells to a target cell concentration ofthe first type of cells.
 4. The method of claim 1 further comprisingalternately opening and closing the flow control device at apredetermined frequency.
 5. The method of claim 4 further comprisingestablishing the predetermined frequency for the duty cycle based on theconcentration of cells in the suspension of biological cells beingseparated.
 6. The method of claim 5 further comprising establishing thepredetermined frequency for the duty cycle as directly proportional tothe concentration of cells in the suspension of biological cells beingseparated.
 7. A system for separating a suspension of biological cellscomprising: a) a fluid circuit comprising separator having a housingwith an inlet for introducing the suspension of biological cells intothe gap, a first outlet for flowing a first type of cells from theseparator and a second outlet for flowing a second type of cells fromthe separator; b) a hardware component comprising a pump for flowing thesuspension of biological cells to the inlet of the separator, at leastone flow control device associated with the first outlet and the secondoutlet of the separator for selectively opening and closing the firstand second outlets so as to permit one of the first type of cells andthe second type of cells to flow out of the separator, and aprogrammable controller configured to operate the pump so as to flow thesuspension of biological cells to the inlet of the separator at apredetermined inlet flow rate and to alternately open and close the flowcontrol device in accordance with a predetermined duty cycle.
 8. Thesystem of claim wherein the programmable controller is configured toalternately open and close the flow control device such that the dutycycle is equal to the ratio of a target flow rate of first type of cellsthrough the first outlet to the predetermined inlet flow rate.
 9. Thesystem of claim 8 wherein the programmable controller is furtherconfigured to determine the target flow rate of the first type of cellsas the product of the inlet flow rate times the ratio of cellconcentration of the suspension of biological cells to a target cellconcentration of the first type of cells.
 10. The system of claim 7wherein the programmable controller is further configured to alternatelyopen and close the flow control device at a predetermined frequency. 11.The system of claim 10 wherein the programmable controller is furtherconfigured to establish the predetermined frequency for the duty cyclebased on the concentration of cells in the suspension of biologicalcells being separated.
 12. The system of claim 11 wherein thepredetermined frequency for the duty cycle is directly proportional tothe concentration of cells in the suspension of biological cells beingseparated.
 13. The system of claim 7 wherein the flow control device isa clamp associated with each of the first and second outlets of theseparator.
 14. The system of claim 7 wherein the flow control device isa two position stopcock associated with both of the first and secondoutlets of the separator.
 15. The system of claim 7 wherein the fluidcircuit comprises spinning membrane separator having a housing and afilter membrane having a first side and a second side, a gap beingdefined between the housing and the first side of the filter membraneand a flow path in fluid communication with the second side of thefilter membrane, an inlet for introducing the suspension of biologicalcells into the gap, a first outlet in communication with the gap forflowing the first type of cells from the separator and a second outletin communication with the second side of the filter membrane for flowingthe second type of cells from the separator.